Therapeutic iron-dextran preparations



United States Patent 3,093,545 THERAPEUTIC IRON-DEXTRAN PREPARATIONSRobert J. Westfall, Kankakee, and Sheldon H. Lewis, Park Forest, Ill.,assignors, by mesne assignments, to Armour Pharmaceutical Company, acorporation of Delaware No Drawing. Filed Aug. 29, 1960, Ser. No. 52,332

3 Claims. (Cl. 16768) strates an average intrinsic viscosity at atemperature of 25 of about 0.025 to 0.25, which complex demonstrates inaqueous solution at an iron concentration of 100 mg. per ml. and atemperature of 25 C. a kinematic Patented June 11, 1963 However, stillbetter results are achieved when the adjustment of the aqueous solutionof iron and dextran to the desired pH is carried out over a period of atleast about one hour.

Funther, it has been discovered that certain of the advantages accruingfrom the therapeutic employment of this iron-dextran complex result fromthe prevention of dextran degradation by pyrolysis during the formationof the iron-dextran complex by the process of this invention.Accordingly, although this object may be accomplished by carrying outthe process at a temperature of less than 70 C., better results areobtained when the formation of the iron-dextran complex is obtained at atemperature of from about to 45 C., and especially desirable in theformation of this improved iron-dextran complex is a temperature of fromabout to C.

viscosity of less than 45 centistokes, which complex is substantiallyfree from dextran degradation products having molecular weights of lessthan about 300, which complex demonstrates on intravenous administrationto mice an LD of at least 1500 mg. Fe per kg., and which complexdemonstrates on intramuscular injection into the thigh of a rabbit at aconcentration of 20 mg. per kg. of body weight less than about 20% ofresidual iron at the injection site after seven days.

This improved iron-dextran product can be prepared by a method whichinvolves forming an aqueous solution having a pH of less than about 2.3of dextran having an average intrinsic viscosity of about 0.025 to 0.25and at least one water soluble ferric salt, which aqueous solution contains less than about 5% (weight/volume) of iron, and adjusting suchaqueous solution to a pH of from 3.5 to 5.0 over a period of at leastabout one-half hour to obtain the iron-dextran complex, provided thatduring the formation of the iron-dextran complex such aqueous solutionis maintained at :a temperature of less than about 70 C.

In obtaining this improved irondextran complex, it is important tocontrol the rate of adjusting the pH of the aqueous solution of iron anddextran to substantially eliminate the formation of undesirableiron-dextran complexes. Although it is especially envisioned that anintervalcontrolled amountbf water-soluble alkali will be combined withthe aqueous solution of iron and dextran by continuous metering toachieve the desired adjustment in the pH thereof to obtain theiron-dextran complex, ithas been found that this improved iron-dextrancomplex may be obtained by a somewhat irregular adjustment of theaqueous solution of iron and dextran to the desired pH, providing thatsuch adjustment in pH is carried out over a period of at least one-halfhour.

On the other hand, it will be appreciated that in order to obtainefficient reaction between the iron and dextran in the formation of thisiron-dext'ran complex the temperature of the aqueous solution of ironand dextran should be maintained at a temperature of at least about 15C.

during the formation of such complex.

As indicated hereinbefore, the formation of this improved iron-dextrancomplex is obtained by adjusting the aqueous solution of iron anddextran having a pH of less than about 2.3 to a pH of from 3.5 to 5.0. IHowever, better results are achieved when such aqueous solution isadjusted to a pH of from about 4.0 to 4.5 in the formation of thisiron-dextran complex. Moreover, especially desirable results areprovided by combining vwith the aqueous solution of iron and dextranhaving a pH of less than about 2.3 about 0.85 to 0.95 equivalent of awater-soluble base per equivalent of ferric anion contained in theaqueous solution of iron and dextran. Accordingly,-in this especialaspect of the invention adjustment of the aqueous solution of iron anddextran to the desired pH is accomplished by combining therewith acontrolled neutralizing amount of base.

There is further contemplated by this invention treatment of the aqueoussolution of the iron-dextran complex obtained by the foregoing processto remove from the aqueous phase thereof the soluble salts formed inthereaction between the Water-soluble ferric salt, and the dextran.Although the separation of these soluble salts from the aqueous solutionof the iron-dextran complex may be obtained by known procedures such asdialysis, especial advantages in connection with the process of thisinvention are provided by a special precipitation method of removingsuch soluble salts to be outlined in detail hereinafter.

In a preferred practice of preparing this improved iron-dextran complex,the dextran is combined in water with at least one ferric salt of astrong univalent acid, for example, ferric salts of such acids ashydrochloric, nitric, perchloric and trichloracetic. Then, the resultingaqueous solution, which demonstrates a pH of less than about 2.3, isalkalinized to the desired pH by combining therewith, on an intervalcontrolled, continuous metering basis over a period of at least one-halfhour, a water-soluble alkali, such as alkali metal hydroxide, ammoniumhydroxide J and tetramethyl ammonium hydroxide, although better resultsare obtained with alkali metal carbonates and bicarbonates. It will beunderstood that the foregoing pH adjustment is carried out at thespecified temperature to substantially eliminate degradation of thedextran by pyrolysis.

After the iron-dextran complex has been formed in aqueous solution bythe foregoing reaction, the soluble salts in the aqueous phase thereofmay be removed by a procedure which involves combining such aqueoussolution with a water-miscible alcohol, preferably ethanol, or acetoneto precipitate in the resulting mixture the iron-dextran complex, andseparating the resulting precipitate from the supernatant liquid bydecantation, filtration or centrifugation.

The irondextran complex substantially free from soluble salts, asobtained by the foregoing process, may be reconstituted in apharmaceutical carrier, especially an aqueous solution, for therapeuticpurposes.

Although it is recognized that the aqueous solution of the iron-dextrancomplex contemplated by this invention does not meet the strictestmodern usage of the term solution in that it is probably separable byultracentrifugation and ultrafiltration, such aqueous solution does meetthe conventional chemical and physical requirements of the solution, andconsequently for the purposes of this invention it is desirable to referto such product as an aqueous solution.

The following examples will serve to further elaborate the specificdetails of this invention.

Example I The following method has been employed in preparing theimproved iron-dextran complex of this invention:

The equipment utilized in this process included a 150 gallon glassedsteel reaction tank (Pfaudler) having a double jacket serviced withsteam and coolant, equipped with a variable speed agitator and anadjustable baffle having three blades. There was vertically suspended inthe tank a pipe having an internal diameter of one inch and having inthe portion thereof inserted in the tank two perforations in the sidesthereof, each perforation having a diameter of 1 mm.; this pipe waspositioned at the periphery of the tank on the side opposite the baffle.This pipe was connected on the outside of the tank, by suitable valves,with a 50 gallon, open-head tank, referred to hereinafter as the sodiumcarbonate head tank, and with a gallon, open-head tank, referred tohereinafter as the distilled water head tank, to provide a gravity-feedsystem from the head tanks into the reaction tank. The bottom of thereaction tank is of conventional inverted conical configuration having apipe leading from its apex through a two inch glass lined valve into aprecipitation tank disposed beneath the reaction tank. The precipitationtank may be of any convenient size but large enough to contain thematerial introduced thereunto. For purposes of the present practice, a250 gallon capacity is adequate.

There was introduced into the reaction tank 72 liters of pyrogen-freedistilled water, 10.03 liters of an aqueous solution of ferric chloridecontaining 19.96% of iron (weight/volume) equivalent to 2000 grns. ofiron, and 18.6 liters of an aqueous solution of 5000 gms. of dextranhaving an average intrinsic viscosity of 0.063.

The sodium carbonate head tank was filled with a 0.30 M aqueous sodiumcarbonate solution. Then, a total of 171.2 liters of the sodiumcarbonate solution was metered into the reaction tank, while maintainingthe aqueous solution of iron and dextran at a substantially constanttemperature and under controlled agitation.

4 The following data was obtained in the course of the reaction:

Increment volume of Reaction carbonate pH of Temperature Agitation time(min. sodium reaction of reaction speed and sec.) solution solutionsolution, C. r.p.m.

added (liters) 0 0 1. 25 31 195 2715. 13. 4 7 6. 7 1.80 31.0 190 7 27 E5 5 E3 6. 7 1. 95 31.5 190 6- 7 i) 6- 7 6. 7 t") 6.7 1.95 32. 5 190 6.7t) 6. 7 6- 7 t) 6.7 2.00 33.0 188 6. 7 6. 7 6.7 2. 10 4. 5 6. 7 2. 1534. 5 185 6. 7 2.15 34. 5 185 6. 7 2. 25 34. 5 185 15.0 2. 6O 34. 5 1851 2.65 34. 5 185 1 2. 75 t) t) 1 2. 1 3.00 34 5 185 1 3. 20 1 3. 50 1 3.75 34. 5 185 1 4. 05 1 4. 40

*Signifies no reading taken.

The resulting aqueous solution of the iron-dextran complex, in theamount of 271.83 liters, was dropped from the reaction tank into theprecipitation tank where to it was added 580 liters of 3A alcohol(methanoldenatured ethanol), While maintaining constant agitation in thetank. In this precipitation there was obtained a fast-settling, mediumcoarse, medium dark brown granules of precipitate. After about 30minutes the precipitate had settled such that the supernatant liquidcould be decanted by pumping from the tank. Then, the precipitateremaining in the tank was washed with 115 liters of a 65% aqueousethanol solution; this washing operation was repeated twice. The washedprecipitate was removed from the precipitation tank and the residualsupernatant liquid was separated therefrom by vacuum filtration.

A portion of the resulting sand was dehydrated and subjected toanalysis. The results demonstrated, on a dry basis, an iron content of30.7%.

This iron-dextran complex was reconstituted in water at a concentrationof mgs. of iron per ml. The following analytical results were obtainedwith the reconstituted product:

Total solids (weight/volume) 30.86%.

Iron (weight/volume) 9.89%.

Chloride (Weight/volume) 1.13%.

Phenol 0.49%.

Kinematic viscosity (centistokes) 5.3.

LD 3500 mg. Fe/kg. Pyrogen +0.2 C.

Rabbit absorption (visual estimate). 20% Fe retained. Physical stabilityat 100 C 56 hours, p.p.t.

It will be apparent that the foregoing product demonstrated a desirablylow viscosity at a high iron concentration, a low toxicity, desirableabsorption properties, and non-pyrogenicity.

There may be employed in preparing this iron-dextran complex any dextranhaving an average intrinsic viscosity of 0.025 to 0.25 which is suitablefor administration to animals and human beings. By dextran is meant themetabolic product of a certain group of bacteria, of which the mostprominent species presently known is LeuconO- stoc mesenteroides.Although the dextran utilized in this process may be advantageously ofthe foregoing average intrinsic viscosity, significantly better resultsare obtained when the dextran has an average intrinsic viscosity ofabout 0.050 to 0.070.

The term kinematic viscosity refers to the measurement of viscosityobtained by the direct reading of a viscosimeter. On the other hand, theterm intrinsic viscosity is a conventional expression of therelationship of concentration and viscosity, while the employment of theterm average intrinsic viscosity is desirable because there is usuallyinvolved in available dext-ran preparations molecules of dextran havingdifferent molecular weights.

The irondextran complex of this invention should contain at least 16% byweight of iron. However, it is especial advantage of this invention thatiron-dextran complexes can be obtained with an iron content of at least26% by weight and even as high :as 34% by weight.

Another especial feature of this invention is the provision ofiron-dextran complexes containing less than 20% by weight of hydroxide,including oxides. Moreover, by the process of this inventioniron-dextr-an complexes can be obtained havingan hydroxide content ofless than 5% by weight, and even products substantially free froml1ydioxide. By hydroxide, for the purposes of this invention, is meantthe amount of material determined by subtracting from the total solidsthe amounts or irondextran and salt present in the iron-dextran complex.

Furthermore, this iron-dextran complex not only is capable of beingreconstituted in aqueous solution at a concentration of 100 mg. of ironper ml. to obtain a product demonstrating a kinematic viscosity of lessthan 45 centistokes, but such iron-dextran complexes can be obtaineddemonstrating a kinematic viscosity of less than 20 centistokes and evenless than centistokes.

As indicated hereinbefore, the inon-dextran complex of this inventiondemonstrates, on intravenous toxicity measurements in mice, an LD of atleast 1500 mg. Fe per kg. However, there has been obtained by thisprocess iron-dextran products having an LD of greater than 2500 mg. Feper kg., and oftentimes in excess of 3000 mg. Fe per kg.

The absorption properties of this iron-dextran complex can be determinedby a method involving the injection of an aqueous solution of theiron-dextr-an complex into New Zealand white male rabbits, in goodhealth, and weighing 1500 to 2500 tgms. In this analytical procedures,the iron-dextnan solution, in the amount of 20 mg. of iron per kg. ofrabbit is injected into the muscle of one of the hind legs of a rabbit.Seven .days after the injection the rabbits are sacrificed, and themuscles into which the irondextnan complex was injected are excised torexamination. The amount of residual iron in the injected muscle, ascompared with the uninjected muscle of the other hind leg, is determinedchemically by the conventional 2,2- bipyridine method. The iron-dextrancomplex of this invention demonstrates, by this analytical procedure,less than 20% of iron at the site of injection after seven days.Moreover, such iron-dextran complexe may be obtained which demonstrateless than of residual iron at the site of injection after seven days,and even as low as 1-2% of residual iron at the site of injection afterseven days Although, in the process of preparing this iron-dextrancomplex, especial advantages are obtained in combining with an aqueoussolution of dextran and a water-soluble ferric salt a suitable alkali toobtain the desired pH adjustment, this iron-dextran complex may beobtained by first combining the water-soluble alkali and the dextran inthe desired amounts, and then combining the watersoluble ferric saltwith the resulting solution.

Moreover, in carrying out the process of this invention it is desirable,in applying the special precipitation procedure for removing solublesalts from the aqueous solution of the iron-dextran complex, to employas the alkali and adjusting pH of the reaction solution, a water-solublealkali which forms, on reaction withthe anion of the ferric salt a saltwhich is soluble in the resulting mixture of alcohol or acetone andwater.

This iron-dextran complex is useful, in humans or in animals, butespecially in domestic animals, either orally or parenterally intherapeutic circumstances in which iron administration is desirable.However, especially desirable results are obtained by the intramuscularor subcutaneous administration of an aqueous solution of thisiron-dextran complex for hematopoietic purposes.

While in the foregoing specification various embodiments of thisinvention has been described in considerable detail for the purpose ofillustration, it will be apparent to those skilled in the art that thisinvention is susceptible to other embodiments and that many of thesedetails can be varied widely without departing from the basic conceptand spirit of the invention.

We claim:

"1. An iron preparation for therapeutic purposes, comprising an aqueoussolution of a complex of iron and dextran, which complex demonstrates anaverage intrinsic viscosity at a temperature of 25 C. of from 0.025 to0.25, which complex demonstrates in aqueous solution at an ironconcentration of mg. per ml. at a temperature of 25 C. a kinematicviscosity of less than 45 centistokes, which complex is substantiallyfree from dextran degradation products having molecular weights of lessthan 300, which complex demonstrates on intravenous administration tomice an LD of at least 1500 mg. Fe per kg., and which complexdemonstrates, on intramuscular injection into the hind leg of a rabbitat a dose concentration of 20 mg. of iron per kg. of body weight, lessthan 20% residual iron at the injection site after seven days.

2. In a method of preparing an iron product for therapeutic purposes,the steps of forming an aqueous solution having a pH of less than about2.3 of dextran having an average intrinsic viscosity of from 0.025 to0.25 and at least one water-soluble ferric salt, and adjusting theresulting aqueous solution to a pH of firom 3.5 to 5.0 over a period ofat least one-half hour to obtain the irondextran complex, whilemaintaining said aqueous solution during the formation of theiron-dextran complex at a temperature of less than 70 C.

3. In a method of preparing an iron product for therapeutic purposes,the steps of combining in water dextran having an average intrinsicviscosity of from about 0.050 to 0.070 and a water-soluble ferric saltof a strong univalent acid, adjusting the resulting aqueous solution toa pH of from 4.0 to 4.5 over a period of at least one hour to obtain theiron-dextran complex, and removing from the resulting aqueous solutionat least a substantial portion of the soluble salts therein.

References Cited in the file of this patent UNITED STATES PATENTS Londonet a1 Apr. 28, 1959 London et al. Jan. 21, 1958 OTHER REFERENCES

1. AN IRON PREPARATION FOR THERAPEUTIC PURPOSES, COMPRISNG AN AQUEOUSSOLUTION OF A COMPLEX IRON AND DESTRAN, WHICH COMPLEX DEMONSTRATES ANAVERAGE INSTRINSIC VISCOSITY AT A TEMPERATURE OF 25*C. OF FROM 0.025 TO0.25, WHIC COMPLEX DEMONSTRATES IN AQUEOUS SOLUTION AT AN IRONCONCENTRATION OF 10 MG. PER ML. AT A TEMPERATURE OF 25*C. KINEMATICVISCOSITY OF LESS THAN 45 CENTISTOKES, WHICH COMPLEX IS SUBSTANTIALLYFREE FROM DEXTRAN DEGRADATION PRODUCTS HAVING MOLECULAR WEIGHTS OF LESSTHAN 300, WHICH COMPLEX DEMONSTRATES ON INTRAVENOUS ADMINISTRATION OFMICE AN LD50 OF AT LAST 1500 MG. FE PER KG., AND WHICH COMPLEXDEMONSTRATES, ON INTRAMUSCULAR INJECTION INTO THE HIND LEG OF A RABBITAT A DOSE CONCENTRATION OF 20 MG. OF IRON PERKG. OF BODY WEIGHT, LESSTHAN 20% RESIDUAL IRON AT THE INJECTION SITE AFTER SEVEN DAYS.
 2. INAMETHOD OF PREPARING AN IRON PRODUCT FOR THERAPEUTIC PURPOSES, THE STEPSOF FORMING AN AQUEOUS SOLUTION HAVNG A PH OF LESS THAN ABOUT 2.3 OFDEXTRAN HAVING AN AVERAGE INSTRINSIC VISCOSITY OF FROM 0.25 TO 0.25 ANDAT LEAST ONE WATER-SOLUBLE FERRIC SALT, AND ADJUSTING THE RESULTINGAQUEOUS SOLUTION TO A PH OF FROM 3.5 TO 5.0 OVER A PERIOD OF AT LEASTONE-HALF HOUR TO OBTAIN THE IRONDEXTRAN COMPLEX, WHILE MAINTAINING SAIDAQUEOUS SOLUTION DURING THE FORMATION OF THE IRON-DEXTRAN COMPLEX AT ATEMPERATURE OF LESS THAN 70*C.